| Hepatic ischemia reperfusion injury(HIRI) is a frequently encountered complication in a variety of clinical scenarios including hepatectomy, liver transplantation, liver trauma, hemorrhagic shock and cardiogenic shock. It is also the most important reason for post-operation liver dysfunction, primary graft failure and a risk factor influencing the long-term graft survival. How to intervene liver failure caused by HIRI and protect the liver attracts more and more global attention. To explore the drug to prevention and control of HIRI has become a research hotspot.In order to reduce HIRI, many scholars have done a lot of researches. They shorten ischemia time, perform ischemia preconditioning and ischemia posttreatment so as to reduce the injury. Exploring the drug to prevent HIRI has become a research hotspot. Drug treatment aims at some mechanism during HIRI, utilizing its pharmacological effects to reduce oxygen free radicals, calcium overloading, inflammatory mediators. Or it takes advantage of certain active substance to enhance the tolerance of liver cell to HIRI directly and indirectly. Thus, injury can be relieved so as to achieve the protection. Erythropoietin (EPO) is a salivary glycoprotein hormone synthesized mainly by the juxtaglomerular cells located in the junction of renal cortex and medulla which can stimulate hematopoiesis in bone marrow. Its primary duty is to promote erythropoiesis.EPO can form EPO-EPOR signaling system through combining with erythropoietin recptor(EPOR) expressed by some kinds of non-erythroid organs, tissues, and cells, such as central nervous system, endothelial cells, uterus. Then it can involve in varieties of non-hematopoietic biological activity. More and more evidences show that EPO has the protective function of organs, tissues and cells. However, this function is independently of its hematopoietic effect.In the process of liver disease treatment, using recombinant human erythropoietin (rHuEPO) treatment in mice and rats, and can significantly reduce the ischemia/reperfusion(I/R) injury, including hepatic histopathological scores, enzymology indexes, apoptosis, harmful cells signal transduction and the function of reactive oxygen species. Decisive pathophysiological mechanisms contributing to the in vivo I/R injury to the liver are mediated or enhanced by endothelial and Kupffer cells, i.e. by liver cells other than hepatocytes rHuEPO may directly or indirectly act on these cells and thus influence the injurious response. Thus, elucidating the protective mechanism of rHuEPO on liver I/R injury is necessary.In this study, we employed liver auto-transplantation rat model to imitate the HIRI process. We discussed the protective effect of rHuEPO against HIRI through studying the changes in liver enzyme indicators, pathological change and cell apoptosisin liver cell and cell apoptosis. We also studied the PI3K signal pathway at gene transcription and protein expression level, maneged to find a possible rHuEPO protection mechanism and also hope to provide some scientific evidence for further understanding of the rHuEPO preconditioned protection. Part1liver auto-transplantation model of ratsPurposeIdeal animal model is the basis of liver transplantation experimental study."double sleeve" rat model raised by Kamada is a mature and wide-used animal model for liver transplantation. However, this model dose not anastomosis liver artery, can not be completely ruled out the immune factors, thus can’t fully represent HIRI pathophysiology. So we choose the HIRI model built in our center to imitate clinical liver transplantation, ruled out the immune and anastomosis liver artery factors. Now we summarize the experience obtained during building the model, management before and after the operation to expect the model has a high success rate and repeatability.Method1. Animals55pure SPF male SD rats were divided into two groups.27of them were trained for early model building. The other28were experimented following the experimental design and drug preconditioning method. Among the28rats group, three were in sham group, the other were equally divided into I/R, I/R+rHuEPO, I/R+DMSO, I/R+rHuEPO+LY294002and I/R+LY294002group, five each.2. Preoperative preparation was done. After the liver and related structureswere fully freed, the rats were heparinized with37.5U/ml heparin. Then4℃lactated Ringer’s solution which contains12.5U/ml heparin were perfused through portal vein and abdominal aorta using double constant pressure cold perfusion. Methods were the same as rat autologous transplantation model except preservation in cold reperfusion and vascular anastomosis operation.3. Observation time and content life quality and survival rate of rats were observed at24h,48h and72h. 4. Statistical analysis SPSS13.0was employed to draw the survival curve.Result1.Warm anhepatice phase was from blocking the PV of heparinized rats to the beginning of cold reperfusion while anhepatic phase lasted until reopening of PV. To standardized research subjects, we set the anhepatic phase to same30min. Wherein warm ischemia time was2.5-3.5min and cold perfusion time was unified to15min.2. Early in the experiment, survival rate of the rats achieves92.31%that met the design requirement. Modeling process was also successful and no modeling-caused death appeared. Studying the impact of experimental drug on the rat72h survival rate, there was no death after42h except the LY294002proconditioned group. In I/R+LY2940002group,72-hour survival rate is low and it could be promoted when combined with rHuEPO.Summaryliver auto-transplantation model simulated the whole clinical liver transplantation process. Thus we can observe HIRI more directly and objectively. When animal model fully met this experimental design, operations followed the primary principle of animal experiment strictly and operators payedattention to proper handling of the details, we can improve the post-operation recovery process significantly and even influence the survival of the rats directly.Part2the protective effect of rHuEPO preconditioning of liver auto-transplantation in ratsPurposeWe managed to observe protective effect of rHuEPO preconditioning through liver auto-transplantation HIRI rat model and preliminarily discussed the protective effect at liver enzyme indicator level, pathomorphology and liver cell apotosis in rats.Method 1. Established liver auto-transplantation rat model.2. Animals60rats were randomly assigned into three groups.Group Ⅰ:Sham group (n=20).Laparotomy and dissociation of the liver only. Group Ⅱ:I/R group (n=20). The rats received1ml saline via the tail vein30min before the operation. Group Ⅲ:I/R+rHuEPO group (n=20). The rats were treated the same as group Ⅱ but received a single intravenous dose of weight(kg)×3000U/kg rHuEPO intravenously via the tail vein30min before the operation. The rHuEPO was diluted in1ml saline.Except sham group, during orthotopic autologous transplantation, all liver all liver went through15min cold reperfusion and30min anhepatic phase.3.Sample collection and managementIn each group,5rats were humanely killed after3,6,12and24h from the beginning of reperfusion.4ml blood samples were collected via the inferior vena cava and then injected into biochemical pipe. Let the blood coagulated under room temperature, centrifuged, then took the upper serum sample to store at-20℃for further analysis.The left lobe of the liver was removed, snap frozen in liquid nitrogen and stored in10%buffered formalin.4. Measurement of Serum ALT, AST, LDH, histopathologic analysis and TUNEL assay to detect the liver cell apoptosis5. Statistical analysis measurement data were presented as mean±standard deviations. The groups were compared using factorial design analysis of variance. A p value<0.5is considered statistically significant. All the analyses are performed using SPSS13.0.RESULT1. Alterations in the serum AST, ALT and LDH levelsThe serum levels of AST were not significantly different among the groups at the same time point (data not shown), but the serum levels of ALT and LDH were significantly different among the groups (P<0.05). The changes in the serum levels of ALT and LDH after the operation, compared with the shamgroup, were significantly increased in I/R+rHuEPO group and were statistically different (P<0.05). However, at the same point after hepatic ischemia reperfusion, serum ALT and LDH levels significantly decreased in I/R+rHuEPO group compared with I/R group and were statistically different(P<0.05).The serum levels of ALT and LDH increased rapidly3hafter reperfusion and reached a peak at6h in all groups and then decreased. It did not come back to common level yet24h after reperfusion and were significantly different compared with sham group(P<0.05).2. Histopathological alterationSectionsof the liver obtained at6h after reperfusion were evaluated for histopathological analysis. The liver tissue in sham group showed basically normal structure. The liver sinusoidals were arranged in order with no neutrophil infiltration. after6h reperfusion, I/R group showed severe swelling of liver cells, ill-liver cells, obvious hepatic lobule central vein and hepatic sinusoid congestion, narrow hepatic sinusoid and disorders liver cells, varying degrees of edema and vocuolar degeneration, possible punctate necrotic. Significant neutrophil aggregation and infiltration can be seen, especially around the central vein.The histological scores for I/R injury were significantly higher than sham groupand were statistically different(P<0.05). However, in I/R+rHuEPO group, the liver congestion got some relief. Hepatic lobule was basically normal.Lobule central vein, hepatic cell cord, liver sinusoids got clear structure. Cell degeneration neutrophil infiltration is not obvious.3. liver cell apoptosisChoose the liver tissue sample which has bear6h HIRI to check. TUNEL positive cells in I/R and I/R+rHuEPO was much larger than sham group(P<0.01). Apoptotic index of I/R+rHuEPO group is significantly lower than I/R group(P<0.01). Few TUNEL positive cell was seen in sham group.SummaryWe established liver auto-transplantation experimental model rats to simulatethe process of HIRI. Changes in enzyme indicator level, pathomorphology level and liver cell apoptosis level indicated that this model represented pathophysiological process of HIRI well. rHuEPO preconditioning can decrease serum ALT and LDH, reduce cell apoptosis and structure damage and express protective function of HIRI caused by transplantation.Part3PI3K/AKT signal pathway in the protective effect of rHuEPO preconditioning against HIRIPurposeOn the basis of Part2, we employed the model the same as Part1and PI3K inhibitor LY294002. We measured the serum enzymology, tissue morphology and the protein expression of AKT and p-AKTser473to discuss whether rHuEPO involved in the protection against HIRI.Method1. Model (similar to Part1)2. Animals Group Ⅰ:Sham group (n=20); Group Ⅱ:I/R group (n=20); Group Ⅲ: I/R+rHuEPO group (n=20); Group Ⅳ:I/R+DMSO group (n=20);GroupⅤ:I/R+LY294002group (n-20);Group Ⅵ:I/R+rHuEPO+LY294002group (n=20). All the disposal is similar to Part1. As Group Ⅰ, Ⅱ and Ⅲ have been done in Part2, we just need to collect the data and analysis.3. Sample collection and reservation (similar to Part2)4. Alterations in serum enzyme and histological score (similar to Part2)5. We employed western blot to analysis the expression of AKT and p-AKTser473in six groups Relative active level of AKT and p-AKTser473is expressed by corresponding protein bar average light intensity value. Activation of AKT/p-AKTser47signal pathway is reflected by the ratio of p-AKTser473/β-actin and AKT/β-actin average value.6. Statistical analysis The statistical method is similar to Part2. All the analyses are performed using SPSS13.0.RESULT1. Effect of LY294002(a PI3K inhibitor) preconditioning on liver enzymesThe serum levels of AST, LDHwere significantly different among group I/R, I/R+LY2940002and Sham at3h,6h,12h and24h after reperfusion(timing begins at the end of anhepatic phase)(P<0.01). After being through HIRI, the serum ALT and LDH of the orthotopic autologous transplantation ratswere significantly different when compared with sham group(P<0.01). Wherein changes in the serum levels of ALT and LDH between I/R+DMSO and Ⅰ/Rgroup is not statistically different(P>0.05). Increment of the serum ALT and LDH level in I/R+LY294002group is much higher than I/Rand I/R+DMSO group at the time point referred before(P<0.05). However, serum levels of ALT and LDH increase rapidly in3hafter reperfusion and reached a peak at6h in I/R+LY294002group and then decreased. It still did not come back to common level until24h after reperfusion and were significantly different compared to sham group (P<0.01)2. Effect of pharmaceuticals pretreatment on the liver tissue morphologyHE staining results suggest:after6h reperfusion, in I/R+DMSO group, liver structure is near-normal. However, hepatic sinusoid congestion and light inflammatory infiltration can be seen. LY294002preconditioning group showed hepatocellularcode damage, vast cell degeneration, dissolved and got high histological score. The score was statistically deferent when compared to I/R+DMSO and I/R+LY294002+rHuEPO group. After6h reperfusion, the SD rats pretreated with I/R+LY294002+rHuEPO showed light cell degeneration, near-normalhepatic lobule, and light neutrophil aggregation and infiltration. But necrosis was also can be seen locally. The structure of necrotic liver cell can not be extinguished. All the results reveal that rHuEPO preconditioning can significantly improve the histology of liver after6h reperfusion. Damage was aggravated in I/R+LY294002group, but can be relieved when combined with rHuEPO.3. Effect of LY294002combined with rHuEPO preconditioning against liver I/R injuryThe serum levels of AST and LDH increased significantly in group I/R+rHuEPO+LY294002when compared with I/R+rHuEPO. It was statistically different(P<0.05). But the serum levels of AST and LDH decreased when compared with I/R+LY294002group at the same time point. It was also statistically different (P<0.05).4. Effect of pharmaceuticals pretreatment on AKT and p-AKTser473rHuEPO preconditioningAfter6h reperfusion, difference of AKT expression among Sham, I/R, I/R+DMSO, I/R+LY29400, I/R+rHuEPO and I/R+LY294002+rHuEPO groups was not statistically significant(P>0.05). Butthe ratio of p-AKTser473and total AKT, p-AKTser473phosphorylation increased significantly after HIRI compared with sham group. And the difference was statistically significant(P<0.05). p-AKTser473increased after rHuEPO preconditioning and when compared with I/R the increment of rHuEPOgroupwasstatistically significant(P<0.05). In I/R+LY294002, we observed LY294002inhibit the activation of p-AKTser473and when compared with I/R group, p-AKTser473decreased obviously. LY294002inhibit the p-AKTser473expression induced by rHuEPO preconditioning after HIRI at the same time. Summary1.Western bloting found that rHuEPO preconditioning induced the p-AKTser473expression increased significantly.Increment of p-AKTser473expression, histological damage and decrement of liver enzyme induced by rHuEPO preconditioning were corresponding to each other.2. LY294002(a specific PI3K inhibitor) inhibit p-AKTser473expression induced by rHuEPO and protective effect of rHuEPO.This experiment through the joint use of rHuEPO and LY294002for rats pretreatment, the research results show that can eliminate rHuEPO preconditioning on liver I/R injury of protection.3. To further support PI3K/AKT signaling pathway activation mediates the rHuEPO pretreatment of liver protection. The protein of p-AKTser473, total AKT protein expression changes confirmed the PI3K/AKT signal pathway involvement in protective effect of rHuEPO preconditioning against HIRI.Part4The role of eNOS in protective effect of rHuEPO preconditioning against HIRIPurposeTo study the PI3K/AKT signal pathway involvement in protective effect of rHuEPO preconditioning, this part we discussed the protective mechanism of rHuEPO againt HIRI though detecting the tissue sample, p-eNOSser1177and eNOS protein expression, transcription level, content of NO and ET-1in serum sample.Method1. Liver tissue and serum sample (collected in part2and part3)2. Transcription level of EPOR and eNOS in the liver sample was detected by qPCR.3. Western blot to detect-protein expression of total eNOS and p-eNOSser1177.4. The serum NO level was detected by Nitrate reduction method. 5. ET-1in the serum sample was detected by biotin double antibody sandwich ELISA method.6. Statistical analysis The statistical method is similar to Part2. All the analyses are performed using SPSS13.0.RESULT1. eNOS mRNA in liver tissue alternation by rHuEPO preconditioningAfter6h reperfusion, eNOS mRNA in I/R group increased significantly compared with Sham group. The difference was statistically significant(P<0.05). eNOS mRNA in I/R+rHuEPO group increased significantly compared with I/R, I/R+DMSO and I/R+LY294002group. The differences were statistically significant (P<0.05). But the difference of eNOS mRNA content between I/R+rHuEPO and I/R+LY294002+rHuEPO group was not significant (P>0.05).2. EROP mRNA in liver tissue alternation by rHuEPO preconditioningAfter6h reperfusion, EROP mRNA increased significantly compared with Sham group. The difference was statistically significant (P<0.05). We found this through real-time PCR. When compared with the HIRI group, we did not find content of eNOS mRNAcan be increased by rHuEPO preconditioning or can be decreased by LY294002.3. Effect of pharmaceuticals pretreatment on eNOS protein expression and p-eNOSSer1177After6h reperfusion were detected by western blot. We compare the ratio of p-Enos Ser1177and total Enos. It turned out to be no significant difference of Enos protein expression among each group (P>0.05).p-eNOSSer1177was seldom expressed in sham group. However p-eNOSSerll77expressed in rHuEPO preconditioning group increased significantly compared with I/R group.The difference was statistically significant (P<0.05). But p-eNOSSer1177did not increase in I/R+LY294002 group.rHuEPO can improve LY294002inhibition of p-eNOSSer1177, we found this when combine use of rHuEPO and LY29400. The alteration trend of p-EnosSer1177in HIRI groups is in accord with alteration trend of AKT及p-AKTser473. These indicated that P13K signal pathway involved in the adjustment of p-eNOSSer1177.rHuEPO can activate phosphorylation of p-AKTser473and p-eNOSSer1177.4. The influence of rHuEPO pretreatment on the serum NO in ratsContent of serum NO in liver tissue sample at3h,6h,12h,24h after reperfusion was detected by Nitric acid enzyme reduction method. NO in HIRI groups decreased significantly when compared with Sham group (P<0.05). rHuEPO preconditioning can increase NO significantly at the same time point.NO in I/R+LY294002decreased significantly and it were statistically different compared with other groups (P<0.05). After6h ischemic reperfusion, NO of the rats in I/R groups decreased to nadir. Then NO increased as time went by. NO of the rHuEPO preconditioning groups was very close to that of Sham group after24h reperfusion.5. Effect of rHuEPO pretreatment on the serum ET-1in ratsContent of serum ET-1in liver serum sample at3h,6h,12h,24h after reperfusion was detected by ELISA method. ET-1in I/R groups increased significantly when compared with Sham group (P<0.05). rHuEPO preconditioning can decreaseET significantly at the same time point.ET in I/R+LY294002increased significantly and it were statistically different compared with other groups (P<0.05). After3h reperfusion, in I/R groups, the serum ET-1increased to a high level. Then the serum ET-1decreased as time went by. The serum ET-1in the rHuEPO preconditioning groups was very close to that in the sham group after24h reperfusion.Summary1. rHuEPO preconditioning does not increase the liver tissues EPOR mRNA levels, but can increase the eNOS mRNA levels. No influence about the EPOR mRNA levels in liver, when combined use of PI3K inhibitor LY294002.2. rHuEPO pretreatment increase the expression of p-eNOSser1177protein and LY294002pretreatment inhibits the expression of p-eNOSser1177protein in liver tissue. It indicates that the PI3K/AKT/eNOS cell signaling pathway is involved in the protective effect in the liver I/R injury.3. Balance between the serum NO and ET-1was broken after HIRI. rHuEPO preconditioning can increase the serum NO significantly and adjust the balance between the serum NO and ET-1. It confirmed the rHuEPO pretreatment by activating p-eNOSser1177increases the release of NO, play on the protective in liver ischemia-reperfusion injury.Conclusion1. liver auto-transplantation model was employed in this experiment.it can simulate pathophysiological process of HIRI, increment of liver enzyme indicator, alteration of liver morphology and occurrence of apoptosis and necrosis also occurred in this model.2. rHuEPO pretreatment can obviously inhibit the liver I/R injury caused by the increase of serum ALT, LDH level, and improve the morphological structure of liver tissue, inhibiting hepatocellular apoptosis and relieve the liver pathological damage.3. Specific PI3K blocker LY294002significantly inhibited the increase of the p-AKTser473expression induced by rHuEPO and its protective effects. These indicated that the rHuEPO preconditioning may protect liver against HIRI through activating PI3K/AKT signal pathway.4. LY294002pretreatment significantly inhibited the increase of rHuEPO preconditioning induced p-eNOSser1177expression in liver tissue. It indicates that the PI3K/AKT/eNOS cell signaling pathway is involved in the protective effect in the liver I/R injury.5. rHuEPO preconditioning increased NO through activating p-eNOSser1177to adjust the ratio of NO/ET-1,protect liver against HIRI. |
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